Synthesis and Purification of Glucuronic Acid Glycosides

Synthesis of the glucuronic acid donor was carried out according to Jongkees and Withers (2011) in a dark environment [18 (link)]. To IXN, in a dry flask in argon atmosphere, 20 mg of IXN was dissolved in dry acetonitrile (dried with molecular sieves, Sigma Aldrich, Saint Louis, MO, USA) followed by the addition of 27 mg (1,2 eq) of acetobromo-α-d-glucuronic acid methyl ester. The reaction was left to stir for 5 min before AgO₂ (2,5 eq, 32.4 mg) was added. The reaction was left at room temperature for 20 h before being filtered through a celite plug, followed by silica gel (mesh 80–200), filtered through filter paper, and concentrated. The reaction product was resuspended in EtOAc and washed with H₂CO₃ twice, with water twice, and with brine once before being concentrated. Deprotection was conducted by the addition of MeOH/H₂O, changing the pH to 12, and leaving the product to stir for a further 3 h, before performing liquid–liquid extraction. Lastly, the obtained suspension was filtered through celite, and the filtrate was columned with Sephadex LH-20 and prepared with preparative HPLC according to previous work [43 (link)]. To obtain 6-PN-7-O-Glc and 8-PN-7-O-Glc, a mixture of 6-PN and 8-PN was used as starting material.